Kidney Week

Abstract: TH-PO488

Proximal Tubule-Specific Regulation of NPT2a by NHERF1

Session Information

• Fluid and Electrolytes: Basic - I
  October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Gagnon, Kenneth, University of Louisville, Louisville, Kentucky, United States

Kenneth Gagnon,

• Barati, Michelle T., University of Louisville, Louisville, Kentucky, United States

Michelle T. Barati,

• Kitterman, Kathleen, University of Louisville, Louisville, Kentucky, United States

Kathleen Kitterman,

• Lederer, Eleanor D., University of Louisville; Robley Rex VA Medical Center, Louisville, Kentucky, United States

Eleanor D. Lederer,

Background

NHERF1 (Na-H Exchanger Regulator Factor 1), a PDZ protein that expresses an Ezrin binding domain (EBD), regulates proximal tubule BBM expression of NPT2a (Na-phosphate cotransporter), a critical determinant of phosphate homeostasis, through interactions with NPT2a and Ezrin (an actin cytoskeleton linking protein). Knockout of NHERF1 or Ezrin results in aberrant trafficking and diminished BBM expression of NPT2a. Additionally, knockout of NHERF1 decreases association of NPT2a with Ezrin. We hypothesize that NHERF1 regulates NPT2a trafficking through PDZ interactions and NPT2a anchoring through EBD interactions.

Methods

To test our hypothesis, we generated cDNA constructs encoding: NPT2a full-length (FL); NPT2a
PDZ1-deficient (TRL-); NPT2a/NHERF1 chimera wherein the EBD of NHERF1 replaces the TRL motif of
NPT2a (Chimera); NHERF1 full-length (FL); and NHERF1 EBD deficient (EBD-). We transfected HEK293 cells, which express Ezrin but not NPT2a or NHERF1, with wild-type and mutant NPT2a and NHERF1 constructs and assessed their expression and function with ³²P-phosphate uptake, immunofluorescence microscopy, and differential centrifugation/western blot experiments.

Results

HEK293 cells expressing NPT2a FL, NPT2a TRL-, or NPT2 Chimera constructs all exhibited 2.5-fold greater phosphate uptake versus sham- or vector-only transfections. Co-transfection with NHERF1 FL or NHERF1 EBD- constructs did not enhance phosphate uptake. Contrary to the diminished NPT2a membrane expression observed in proximal tubule cells lacking NHERF1 or transfected with NPT2a TRL-, immunofluorescence of HEK293 cells transfected with wild type and mutant NPT2a constructs demonstrated plasma membrane localization in the absence or presence of NHERF1. Wild type and mutant NHERF1 constructs exhibited global expression throughout the cell, even at the lowest transfection level (75 ng/ul). Differential centrifugation/Western blotting of transfected cells revealed wild type and mutant NPT2a expression in only the organelle and plasma membrane compartments, while wild type and mutant NHERF1 construct expression was observed in organelle, plasma membrane, and cytosol compartments.

Conclusion

These studies suggest that 1) NHERF1 regulation of NPT2a is proximal tubule specific; and 2) unidentified accessory protein(s) are essential for the BBM trafficking, localization, and function of NPT2a in proximal tubule.

Funding

• Veterans Affairs Support